STEVE IAN. PERLMUTTER - Primate Testing - 2006

Abstract: DESCRIPTION (provided by applicant):
The proposed experiments are components of a research effort whose
long-term objective is to understand the role played by the spinal cord
in the control of voluntary movements of the primate arm. This goal
includes a search for therapies or interventions that will overcome the
motor deficits associated with spinal cord injury. The proposed study
advances these goals by elucidating the functional organization of
spinal interneurons controlling forearm movements in the normal,
behaving monkey. A thorough understanding of normal spinal function is
essential before studies of motor impairment and recovery following
injury can be interpreted usefully.

The specific aims of the project are: 1. What is the role of
propriospinal interneurons located rostral to the cervical enlargement
in the control of primate arm and hand movements? 2. How are motoneuron
and interneuron excitability regulated during normal movements by
inhibitory mechanisms in the spinal cord? 3. Is the normal activity of
spinal motoneurons during voluntary movement dependent on the action of
monoamines? The activity of cervical neurons will be recorded during
voluntary reaching, isolated wrist movements, and cocontraction of
flexor and extensor muscles of the wrist in the awake monkey. Inhibitory
and neuromodulator inputs to spinal neurons will be manipulated with
local iontophoresis of pharmacological agents that activate or block
serotonin, noradrenaline, GABA or glycine receptors. Input and output
connections of interneurons will be identified with spike-triggered
averages of EMG and natural and electrical stimulation of peripheral
afferents and descending pathways. These studies will elucidate the
mechanisms by which the excitability of interneurons and motoneurons are
controlled during voluntary movements and lay the foundation for future
studies on the mechanisms of motor dysfunction and recovery from injury.

Activity of Spinal Interneurons and Their Effects on
Forearm Muscles During Voluntary Wrist Movements in the Monkey

Steve I. Perlmutter, Marc A. Maier, and Eberhard E. Fetz

During training and recording sessions, the monkeys sat upright in a
primate chair adjusted for each animal. The working arm was restrained
with the elbow bent at 90°. The hand was held with the fingers straight
and the wrist in the midsupination/pronation position. The
flexion/extension axis of the wrist was aligned with the shaft of a
torque transducer. The other arm was restrained loosely.

Behavioral paradigm
The monkeys were trained to generate isometric, ramp-and-hold flexion
and extension torques about the wrist. Torque controlled the position of
a cursor on a video screen in front of the animal. To obtain a fruit
juice or applesauce reward, the monkeys held the cursor for 1.5-2 s
within a target window that specified a given flexion or extension
torque 0.2 N-m. For animals B and R, the target windows alternated back
and forth between fixed flexion and extension levels. These animals
produced ramp-and-hold torques in each direction starting from a
maintained torque in the opposite direction (Fig. 1A). Monkey W began
each trial by positioning the cursor within a center target window,
corresponding to zero torque, for 1-4 s. Then a target window appeared
at one of six positions randomly selected from three flexion and three
extension levels. Monkey W produced ramp-and-hold torques in each
direction starting from rest

Surgical implants
All surgeries were performed with the use of aseptic techniques with the
animals under 1-1.5% halothane or isoflurane anesthesia after training
was completed. Atropine sulfate was administered preoperatively;
antibiotics (cefazolin, 25 mg/kg) and analgesics (ketoprofen, 5 mg/kg)
were given postoperatively. Head stabilization lugs were cemented to the
exposed skull with dental acrylic, which was anchored to the bone with
screws.

A stainless steel recording chamber was implanted over the lower
cervical spine (Fig. 2). Following a midline incision, soft tissue was
retracted to expose the lateral masses of the midcervical to upper
thoracic vertebrae. The dorsal spinous processes of the C4-T2 vertebrae
were removed and a unilateral laminectomy of C5-T1 was performed. Each
lamina was removed with a rongeur from its junction with the facet to
just past the midline. Bone screws were introduced into the vertebrae,
and the recording chamber was positioned over the laminectomy and
cemented in place with dental acrylic. The skin and underlying soft
tissue were pulled tightly in layers around the chamber with
purse-string sutures. The outer surface of the skin was held in contact
with the underside of a small flange near the top of the chamber,
protecting the exposed skin margin. The chamber was closed at all times
with a protective cap except during recording sessions.

In monkeys B and R, vitalium screws were inserted into the intact
C4-T2 laminae and the implant remained stable for ~2 mo. In monkey W,
the stability of the implant was extended to 6 mo with the surgical
technique of Anderson et al. (1991) (Fig. 2).

The implant procedure fused the C4-T2 vertebrae. After recovery from
surgery, the monkeys exhibited a stiffened posture of the upper back,
but showed no signs of discomfort nor any neurological symptoms. The
animals' behavior in their home cages returned to near normal and their
performance at the trained task quickly reached preoperative levels.

Bipolar electromyographic electrodes were implanted in 10-14 forearm
muscles. In monkey W, patch electrodes (Microprobe, Clarksburg, MD) and
multistranded stainless steel wires were sutured to surgically exposed
muscles with the monkey under isoflurane anesthesia. Connecting wires
were led subcutaneously to a multicontact socket connector cemented to
the skull. In monkeys B and R, wire pairs were inserted transcutaneously
with the animals under ketamine anesthesia; external wires and
connectors were taped to the upper arm and concealed in a jacket worn by
the monkey. Replacement of transcutaneous electrodes every 2-4 wk
ensured recording quality.

Recording procedure
During recording sessions, the head and vertebral implants were secured
to the primate chair with nylon screws. The implants were held firmly,
but both restraints were somewhat flexible, allowing the animal to make
small postural adjustments. Monkey B received occasional intramuscular
injections of diazepam (2-4 mg) to eliminate excessive movements that
jeopardized stable neuronal recordings. These procedures were well
tolerated: all monkeys moved voluntarily from their home cages and
seated themselves in the primate chair for each day's session.

An X-Y positioning stage and microdrive were mounted on the chamber
(Fig. 2). Activity of neurons in the C6-T1 spinal segments was recorded
extracellularly with glass-insulated tungsten or Elgiloy electrodes
advanced through the dura mater under direct visual observation through
a dissecting microscope. Neurons were isolated while the monkey
performed the isometric wrist task for 2-5 h/day. Stable recordings of
individual neurons could be maintained for >30 min when the monkey sat
quietly. As many as 100 tracks were made in each animal during a period
of 2-6 mo. None of the animals exhibited observable behavioral deficits
at any time.

Rats, mice, birds, amphibians and other animals have
been excluded from coverage by the Animal Welfare Act. Therefore research
facility reports do not include these animals. As a result of this
situation, a blank report, or one with few animals listed, does not mean
that a facility has not performed experiments on non-reportable animals. A
blank form does mean that the facility in question has not used covered
animals (primates, dogs, cats, rabbits, guinea pigs, hamsters, pigs,
sheep, goats, etc.). Rats and mice alone are believed to comprise over 90%
of the animals used in experimentation. Therefore the majority of animals
used at research facilities are not even counted.